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Misfolded proinsulin accumulates in expanded pre-Golgi intermediates and endoplasmic reticulum subdomains in pancreatic beta cells of Akita mice ¹

Division of Cell and Molecular Pathology, Department of Pathology, University of Zurich, Zurich, Switzerland

²Correspondence: Division of Cell and Molecular Pathology, Department of Pathology, University of Zurich, Schmelzbergstrasse 12, CH-8091 Zurich, Switzerland. E-mail: juergen.roth{at}usz.ch

SPECIFIC AIMS

Disorders due to incorrect protein folding constitute a large and diverse family of diseases. Our aim was to characterize and quantify by electron microscopic morphometry and 3-D reconstructions the changes in the early secretory pathway of pancreatic ß cells of Akita mice in which misfolded proinsulin is synthesized and to identify subcellular site(s) of accumulation of proinsulin by immunogold labeling.

PRINCIPAL FINDINGS

1. Dilated rough endoplasmic reticulum subdomains are significantly increased in pancreatic ß cells of Akita mice
The rough endoplasmic reticulum (ER) constitutes a site of quality control of protein folding. In Akita mice pancreatic ß cells, volume density of the entire rough ER increased 1.7-fold over that of C57Bl/6J control (Fig. 1 ). This was due to a 2.9-fold increased volume density of dilated ER subdomains which then corresponded to 75% of total rough ER in Akita mice ß cells. Dilated ER subdomains also existed in control mice ß cells (Fig. 1) , which demonstrated that it was this portion of the ER which was selectively increased. Volume density of transitional ER was increased 2.75-fold in Akita mice ß cells.

View larger version (217K): [in this window] [in a new window]   Figure 1. Fine structural details of ß cells of islets of Langerhans from control and Akita mice. A) In control mice ß cells, narrow cisternae of rough endoplasmic reticulum (RER) are continuous (arrowheads) with dilated portions of cisternae (RER*). B) A field of cytoplasm with rough endoplasmic reticulum (RER) and a transitional element (TE) of RER, a Golgi associated pre-Golgi intermediate (pGI), the Golgi apparatus (G) and mature secretory granules (SG). C, D) In Akita mice, ß cells contain abundant dilated rough endoplasmic reticulum that is continuous with narrow cisternal portions (arrows in panel C), and enlarged pre-Golgi intermediates (pGI in panel D). Scale bars = 0.21 µm (A); 0.17 µm (B, D); 0.27 µm (C).

2. Pre-Golgi intermediates of Akita mice ß cells are greatly expanded
Our previous studies of subcellular distribution of key components of protein quality control machinery in normal cells and tissues suggested involvement of pre-Golgi intermediates. In Akita mice ß cells, pre-Golgi intermediates appeared as prominent structures (Fig. 1) . As deduced from analysis of series of consecutive ultrathin sections, Golgi-associated and peripheral pre-Golgi intermediates consisted of an average of 11 tubules (range 5–15 tubules) with an average diameter of 102 nm (range 70–135 nm) and a length of up to 500 nm. In control mice ß cells intermediates were composed of an average of 3.6 tubules (range 2–5 tubules) with a length of up to 500 nm. Tubules were always closely related to, but discrete from, transitional elements of rough ER. Volume density of Golgi-associated and peripheral pre-Golgi intermediates in Akita mice was increased 5.8-fold and 4.5-fold, respectively (see Fig. 3 ). Increased volume density was reflected in an almost 2-fold increase of the average diameter of pre-Golgi intermediates based on 3-D reconstruction. This indicates that their increased volume density is due to an increase in size because of the higher number of tubules per pre-Golgi intermediate. As deduced from analysis of series of consecutive ultrathin sections, Golgi-associated and peripheral pre-Golgi intermediates in Akita mice ß cells consisted mainly of slightly curved tubules.

View larger version (40K): [in this window] [in a new window]   Figure 3. Schematic representation of the secretory pathway of a pancreatic ß cell. The morphometric analysis of narrow (ER) and dilated (ER*) endoplasmic reticulum, pre-Golgi intermediates (pGI) and Golgi apparatus related the major increase in volume density (Vv) to the pre-Golgi intermediates that also contained 40% of labeling for C-peptide (LI: gold particles/organelle). The number of insulin secretory granules (SG) was the same in control and Akita mice ß cells, although they were significantly smaller in the latter.

3. C-peptide accumulates in dilated ER subdomains and pre-Golgi intermediates
In both control and Akita mice ß cells, immunogold labeling for C-peptide was detectable over rough ER and its transitional elements (pre-Golgi intermediates) throughout the Golgi cisternal stack (immature and mature secretory granules) (Fig. 2 ). Based on known proinsulin to insulin conversion in immature secretory granules, immunogold labeling for C-peptide detectable in the ER and up to the Golgi apparatus reflected proinsulin immunoreactivity, whereas immunolabeling over immature and mature secretory granules represented free C-peptide. To obtain a relative total quantitative measure for C-peptide immunolabeling along the secretory pathway, we weighed labeling density (gold particles/µm²) according to volume density of respective organelle. An X² test demonstrated that distribution of immunolabeling in the ß cell secretory pathway was significantly different between Akita and control mice (X²= 29.97, P<0.001). The major contribution to the difference in immunolabeling distribution came from pre-Golgi intermediates. In Akita mice ß cells, 40% of immunolabeling could be assigned to them as compared with 20% in controls. This demonstrated that proinsulin mainly accumulated in expanded pre-Golgi intermediates. Labeling intensity of transitional elements of ER was 0.4% in control and 0.7% in Akita mice ß cells.

View larger version (183K): [in this window] [in a new window]   Figure 2. Distribution of C-peptide in the secretory pathway of control and Akita mice. A) In control mice ß cells, gold particle labeling can be observed over rough ER (RER), pre-Golgi intermediate, Golgi apparatus (G), partially clathrin-coated immature secretory granules (arrows), and mature secretory granules (SG). B–D) In Akita mice ß cells, narrow endoplasmic reticulum (RER in panel C) and dilated one (RER* in panels C, D), enlarged Golgi-associated (pGI in panel B) and peripheral (pGI in panel C) pre-Golgi intermediates as well as the Golgi cisternal stack (G) and secretory granules (SG in panels B, D) are labeled. Bars = 0.31 µm (A); 0.24 µm (B); 0.17 µm (C); 0.3 µm (D).

4. Formation of insulin secretory granules in Akita mice is not impaired
Mice have two nonallelic insulin genes. Since the mutation in Akita mice affects only one allele of the insulin 2 gene, formation of insulin secretory granules occurred. The number of secretory granules (numerical density per unit volume) and their structural features in Akita mice were not different from that of control mice (Fig. 3 ). This demonstrated that formation of secretory granules was not influenced by the presence of misfolded proinsulin. However, their volume density and average volume were reduced to 20% of control mice secretory granules and their average diameter to 58% indicating lack of cargo.

CONCLUSIONS

A missense mutation of the insulin 2 gene (Cys96Tyr) in Akita mice disrupts one of two inter-chain disulfide bonds and results in intracellular accumulation of misfolded proinsulin. We showed that in this animal model of a protein folding disease, pre-Golgi intermediates were most notably enlarged and dilated ER subdomains and transitional ER to a lesser extent. By quantitative immunogold labeling we demonstrated that pre-Golgi intermediates were the major sites of proinsulin accumulation and not the ER. Our morphometric analysis of insulin secretory granules indicated that they were formed at the same scale in both Akita and control mice ß cells, although they were smaller in Akita mice. This indicated that accumulated misfolded proinsulin did not cause a general functional impairment of the early secretory pathway in Akita mice ß cells. Together, first systematic and quantitative analysis of organelle changes in an animal model of a protein folding disease demonstrated involvement of pre-Golgi intermediates and ER subdomains and provided insights into the subcellular pathology of protein folding diseases.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1210fje;

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